1. Field of the Invention
The invention concerns a method for immobilizing biomolecules to a carrier material, a carrier material functionalized with a biomolecule as well as the use thereof.
2. Description of Related Art
The binding of biomolecules, particularly proteins or peptides, but also complex molecules with peptide structural units and amino acids to carrier materials, the so-called solid phase, is known for pursuing different objectives. The carrier materials are insoluble under the specified, usually aqueous, solution conditions. Such a carrier material functionalized with a biomolecule is used in order to fix, concentrate, and/or separate other biomolecules, usually also amino acids, peptides, proteins or complex molecules or supramolecular systems with peptidic structural units. According to the concept of the antigen-antibody reaction, these molecules form specific and structurally-selective complexes with the immobilized biomolecule with the formation of non-covalent van-der-Waals bonds and hydrogen bonds. Thus, peptide units and peptidic secondary structural elements are recognized as receptor units (U. Diederichsen, et al., Bioorganic Chemistry, Wiley Publishing Co. Chemistry, Weinheim 1999, pp. 221 ff; G. Gauglitz et al., Proc. SPIE 4205: Advanced Environmental and Chemical Sensing Technology, 2000, 10).
The biomolecule can be immobilized on the carrier material by physical adsorption, whereby the binding forces that participate are primarily hydrogen bonds and van-der-Waals forces, or by ionic bonding based on electrostatic forces or by covalent bonding by chemical reaction. The covalent immobilization of biomolecules is usually conducted on polymeric carrier materials or carrier materials which have a polymeric surface. For covalent bonding, it is also known to provide the biomolecule with functional groups that can be readily activated, which can enter into a chemical bond with the carrier material. By introducing the energy necessary for the chemical reaction by heating or exposing with energy-rich radiation, reactive intermediates are formed from the functional group of the biomolecule, which then react with the polymer surface and immobilize the amino acid, the peptide, the protein or the complex molecule with peptidic structure by way of covalent bonding.
In this context, WO 91/16425 as well as U.S. Pat. No. 5,853,744 describe the use of azide-derivatized biomolecules, which form nitrenes with cleavage of nitrogen upon heating or by photolysis, which in turn react with polymer surfaces particularly by combined abstraction and insertion reactions and [are] immobilize[d] together with the biomolecule as an amine. WO 91/16425 as well as Clemence et al. (Bioconjugate Chem., 1995, 6, 411-417) describe further the derivatising of biomolecules with diaziridine functions, which covalently bind to the polymer surface after activation and subsequent three-ring opening. In addition, EP 562,373 A discloses the use of epoxy-derivatized polymeric carriers for covalent bonding of biochemical substances.
It is also known to use the sensitizer capacity of benzophenone structures relative to the photochemical H or proton abstraction (both H+ as well as H°) with subsequent recombination of the intermediate radicals for immobilizing amino acids and peptides. In this way, the receptor units (amino acid, peptide) to be immobilized are additionally provided with a benzophenone structure. When this conjugate is photochemically excited in the vicinity of a polymer surface, H or proton abstraction (both H+ as well as H°) from the polymer or from the peptide is bound by the excited benzophenone results, with the formation of radicals on the part of the polymer and/or the peptide, which then enter into combination reactions. In the course of these radical combinations, the functionalized receptor unit to be bound to the polymer is at least partially covalently bound to the surface of the polymer and thus the biomolecules is immobilized (M. Ulbricht et al., J. Membr. Sci. 1996, 120, 239-259; U.S. Pat. No. 5,071,529; J.-F. Clemence et al., Bioconjugate Chem. 1995, 6, 411-417).